Fig. 4.

UHRF1 S652 phophorylation disrupts interaction with USP7 and decreases UHRF1 stability. (A) Superimposed ITC enthalpy plots for the binding of SpacerUSP7 560–664 (syringe) with UHRF1 600–687 and mutations of UHRF1 S652D (Cell). The estimated binding affinity (Kd) numbers are listed in the inset. (B) Stable cell lines were established that express either FLAG-tagged, wild-type or S652 mutants (S652A and S652D). Cells were treated with 50 ug/mL cycloheximide (CHX) as indicated. Extracts were prepared at the indicated time points (top) and used for Western blotting. The amount of UHRF1 was normalized against the corresponding actin signal and the quantitations were shown at the bottom of each panel. (C) HCT116 p53^-/- cells were treated as in Fig. S4D. The extracts were immunoprecipitated with a UHRF1 antibody, followed by Western blotting using USP7 and UHRF1 antibody, respectively. (D) HCT116 p53^-/- cells were treated as in Fig. S4D and released into 50 μg/mL cycloheximide (CHX) containing culture as indicated. Lysates were analyzed by Western blot with UHRF1 and actin antibodies, respectively. UHRF1 band intensity was normalized against the internal actin controls. (E) HCT116 p53^-/- stable cell lines were established that coexpress control or UHRF1 shRNA with indicated Flag-tagged UHRF1 (wild type or mutant). They were seeded at 1 × 10⁴ cells in triplicate 60 mm plates. Cells were trypsinized and counted at indicated time points. Standard deviation bars were obtained from the triplicate counts. (F) A working model. At the G1 and S phases of the cell cycle, UHRF1 level is regulated by the balance of its ubiquitylation and deubiquituylation (mediated by USP7), with deubiquitylation inhibiting UHRF1 proteasomal degradation. CDK1-cyclin B mediates phosphorylation of UHRF1 at M phase, which disrupts the interaction with USP7 leading to an increased turnover and thus reduces steady state levels of UHRF1.